A scanning electron microscope (SEM) has been widely used not only for morphological observation on an inorganic material sample or an organic material sample but also for observation on a biological sample, and is an apparatus suitable for observing microorganisms, such as bacteria and viruses, which cannot be observed by the unaided eye.
Unfortunately, there are problems in that such a biological sample is susceptible to damage due to electron beam irradiation and it is difficult to acquire a high contrast image. For SEM observation on a biological sample, typically, a sample as an observation target is pretreated; in this pretreatment, the sample is fixed with formaldehyde or the like, the surface of which is coated with gold, platinum, carbon or the like or stained with heavy metal or the like. This pretreatment is elaborated to reduce the damage from the electron beam to the sample while improving the contrast.
Recently, a method has also been developed that acquires a high contrast image without the aforementioned coating or staining on a biological sample (see Patent Literature 1 and Non Patent Literature 1). In this method, an observation target sample is attached to the undersurface (back surface) of a thin sample supporting film (carbon film), and irradiated with an electron beam accelerated onto the top surface (front surface) of the sample supporting film at a relatively low voltage. The electron beam incident on the sample supporting film is diffused and spreads in the supporting film. Electrons, reaching the undersurface of the supporting film and therearound, cause secondary electrons to be emitted. The secondary electrons are absorbed by the observation target sample attached to the undersurface of the supporting film to thereby provide contrast, which enables a sharp SEM image to be acquired.
The energy of such secondary electrons is significantly low, which is about 10 eV. The electrons cause almost no damage even if being absorbed by a biological sample. Furthermore, the degree of absorption of the electron beam as it is represented as contrast. Accordingly, a significantly clear and high contrast SEM image can be acquired. Such an observation condition is referred to as an “indirect secondary electron contrast condition”.
The method has further advanced, and methods have been developed that form a conductive thin film under an insulative thin film, and use a charging effect resulting from incidence of an electron beam to further improve resolution and contrast (see Patent Literature 2 and Non-Patent Literature 2).